Abstract
Human β-defensin (HBD) has been recognized as a promising antimicrobial agent due to its broad-spectrum antimicrobial activity against various pathogens. In our previous work, we engineered a chimeric human β-defensin, designated H4, by fusing human β-defensin 3 and human β-defensin 4, resulting in enhanced antimicrobial activity and salt stability. However, the high cost of chemical synthesis due to the relatively large number of amino acids in H4 has limited its applications. To reduce production costs, we aimed to develop an alternative method using a prokaryotic expression system. We first optimized the codon usage of the H4 gene for prokaryotic expression and then cloned it into the pET32a vector, incorporating thioredoxin and enterokinase cleavage sites to minimize toxicity in host cells. The resulting plasmid was transformed into E. coli BL21, yielding a fusion protein (TrxA-EK-H4). Correct cleavage of TrxA-EK-H4 required the addition of urea as a denaturant in the dialysis buffer. However, on-column enzymatic cleavage obviated the need for denaturants and yielded higher-purity rH4. The antibacterial activity of rH4 against multidrug-resistant Acinetobacter baumannii was comparable to that of chemically synthesized H4. This study demonstrates a valuable strategy for efficient purification of challenging proteins and has significant implications for future biotechnological applications.